Method for Purifying Antigen of Foot and Mouth Disease Inactivated Virus by Hydrophobic Interaction Chromatography
A hydrophobic interaction chromatography, virus antigen technology, applied in the preparation methods of peptides, virus peptides, chemical instruments and methods, etc., can solve the problems of death, poor safety, strong side reactions, etc. The effect of high recovery rate
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Embodiment 1
[0028] Get 1000mL of the cell culture supernatant containing FMD virus, the total protein concentration is 0.47g / L, and the FMD virus antigen concentration is 2.1 μg / mL.
[0029] Use a plate ultrafiltration membrane (Sartorius) with a molecular weight cut-off of 300kDa to concentrate the cell supernatant to about 20mL once, and the protein concentration in the sample is about 100μg / mL; the membrane flow rate during ultrafiltration is 100cm / s, and the pressure is controlled below 0.3MPa .
[0030] In the supernatant, adjust the conductivity and pH to above 100mS / cm and pH 8.0 respectively with ammonium sulfate and sodium hydroxide solution in turn, and then feed into the sodium phosphate buffer that has been adjusted to conductance 100mS / cm with ammonium sulfate in advance (pH8.0) balanced Butyl Sepharose 4FF hydrophobic chromatography column (GE Healthcare, 5cm×1.6cm I.D.), after feeding, after continuous elution, ammonium sulfate was used to adjust to sodium phosphate buffer ...
Embodiment 2
[0034] Get 100mL of cell culture supernatant containing FMD virus, the total protein concentration is 0.47g / L, and the FMD virus antigen concentration is 2.1 μg / mL.
[0035]In the supernatant, adjust the conductivity and pH to above 100mS / cm and pH 7.2 respectively with ammonium sulfate and sodium hydroxide solution in turn, then feed into the sodium phosphate buffer that has previously adjusted the conductivity to 100mS / cm with ammonium sulfate (pH7.2) balanced Butyl Sepharose 4FF hydrophobic chromatography column (GE Healthcare, 5cm×1.6cm I.D.), after feeding and continuing to rinse, use ammonium sulfate to adjust the conductivity to 5-100mS / cm sodium phosphate buffer solution (pH 7.2) to carry out gradient elution, and collect the eluate containing the ultraviolet absorption peak whose recovery rate of foot-and-mouth disease virus antigen is greater than 20%. The chromatography medium was regenerated with 1M sodium hydroxide solution.
[0036] The antigen concentration and...
Embodiment 3
[0038] Get 100mL of cell culture supernatant containing FMD virus, the total protein concentration is 0.47g / L, and the FMD virus antigen concentration is 2.1 μg / mL.
[0039] Concentrate the cell supernatant to about 10 mL at a time using a plate-type ultrafiltration membrane (Sartorius) with a molecular weight cut-off of 50 kDa, and the concentration of FMD virus antigen in the sample is about 20 μg / mL; the membrane flow rate during ultrafiltration is 100 cm / s, and the pressure is controlled at 0.3 Below MPa.
[0040] In the supernatant, use ammonium sulfate and sodium hydroxide solution to adjust the conductivity and pH to more than 50mS / cm and about pH 9.0 respectively, then add 10ml of sodium phosphate buffer ( pH 9.0) balanced Phenyl Sepharose 6FF hydrophobic medium, stirred and adsorbed at 100-200rpm for 30min, then added to a hydrophobic chromatography column (GE Healthcare, 5cm×1.6cm I.D.), after continuous elution, adjusted with ammonium sulfate to The sodium phosphat...
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